The study of protein structure and function has historically relied upon the properties and reaction chemistries that are available using the reactive groups of the naturally occurring amino acids. Unfortunately, every known organism, from bacteria to humans, encodes the same twenty common amino acids (with the rare exceptions of selenocysteine (see, e.g., A. Bock et al., (1991), Molecular Microbiology 5:515-20) and pyrrolysine (see, e.g., G. Srinivasan, et al., (2002), Science 296:1459-62). This limited selection of R-groups has restricted the study of protein structure and function, where the studies are confined by the chemical properties of the naturally occurring amino acids, e.g., the natural amino acids limit the ability to make highly targeted protein modifications to the exclusion of all other amino acids in a protein. Additionally, the natural amino acids are limited in their functional activities, e.g., fluorescence, metal chelating, redox-potential, photocaging, etc.
Most modification reactions currently used in the art for the selective modification of proteins involve covalent bond formation between nucleophilic and electrophilic reaction partners that target naturally occurring nucleophilic residues in the protein amino acid side chains, e.g., the reaction of α-halo ketones with histidine or cysteine side chains. Selectivity in these cases is determined by the number and accessibility of the nucleophilic residues in the protein. Unfortunately, naturally occurring proteins frequently contain poorly positioned (e.g., inaccessible) reaction sites or multiple reaction targets (e.g., lysine, histidine and cysteine residues), resulting in poor selectivity in the modification reactions, making highly targeted protein modification by nucleophilic/electrophilic reagents difficult. Furthermore, the sites of modification are typically limited to the naturally occurring nucleophilic side chains of lysine, histidine or cysteine. Modification at other sites is difficult or impossible.
What is needed in the art are new strategies for incorporation of unnatural amino acids into proteins for the purpose of modifying and studying protein structure and function, where the unnatural amino acids have novel properties, e.g., biological properties, not found in the naturally occurring amino acids. There is a considerable need in the art for the creation of new strategies for protein modification reactions that modify proteins in a highly selective fashion, and furthermore, modify proteins under physiological conditions. What is needed in the art are novel methods for producing protein modifications, where the modifications are highly specific, e.g., modifications where none of the naturally occurring amino acids are subject to cross reactions or side reactions. Novel chemistries for highly specific protein modification strategies can find a wide variety of applications in the study of protein structure and function.
One strategy to overcome these limitations is to expand the genetic code and add amino acids that have distinguishing physical, chemical or biological properties to the biological repertoire. This approach has proven feasible using orthogonal tRNA's and corresponding novel orthogonal aminoacyl-tRNA synthetases to add unnatural amino acids to proteins using the in vivo protein biosynthetic machinery of a host cell, e.g., the eubacteria Escherichia coli (E. coli), yeast or mammalian cells. This approach is described in various sources, for example, Wang et al., (2001), Science 292:498-500; Chin et al., (2002) Journal of the American Chemical Society 124:9026-9027; Chin and Schultz, (2002), ChemBioChem 11:1135-1137; Chin, et al., (2002), PNAS United States of America 99:11020-11024; and Wang and Schultz, (2002), Chem. Comm., 1-10. See also, International Publications WO 2002/086075, entitled “METHODS AND COMPOSITIONS FOR THE PRODUCTION OF ORTHOGONAL tRNA AMINOACYL-tRNA SYNTHETASE PAIRS;” WO 2002/085923, entitled “IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS;” WO 2004/094593, entitled “EXPANDING THE EUKARYOTIC GENETIC CODE;” WO 2005/019415, filed Jul. 7, 2004; WO 2005/007870, filed Jul. 7, 2004; and WO 2005/007624, filed Jul. 7, 2004.
There is a need in the art for the development of orthogonal translation components that incorporate unnatural amino acids into proteins, where the unnatural amino acids can be incorporated at a defined position, and where the unnatural amino acids impart novel biological properties to the proteins in which they are incorporated. There is also a need to develop orthogonal translation components that incorporate unnatural amino acids with novel chemical properties that allow the amino acid to serve as a target for specific modification to the exclusion of cross reactions or side reactions with other sites in the proteins. There is also a particular need for protein expression systems that have the ability to produce proteins containing unnatural amino acids in significant quantities that permit their use in therapeutic applications and biomedical research. The invention described herein fulfills these and other needs, as will be apparent upon review of the following disclosure.